Peroxycarboxylic acid compositions can be effective antimicrobial agents. Methods of using peroxycarboxylic acids to clean, disinfect, and/or sanitize hard surfaces, textiles, meat products, living plant tissues, and medical devices against undesirable microbial growth have been described (U.S. Pat. No. 6,545,047; U.S. Pat. No. 6,183,807; U.S. Pat. No. 6,518,307; U.S. Patent Application Publication No. 2003-0026846; and U.S. Pat. No. 5,683,724). Peroxycarboxylic acids have also been used in a various bleaching applications including, but not limited to, wood pulp bleaching/delignification and laundry care applications (European Patent 1040222B1; U.S. Pat. Nos. 5,552,018; 3,974,082; 5,296,161; and 5,364,554). The desired efficacious concentration of peroxycarboxylic acid may vary according to the product application (for example, ca. 500 ppm to 1000 ppm for medical instrument disinfection at neutral pH, ca. 30 ppm to 80 ppm for laundry bleaching or disinfection applications) in 1 min to 5 min reaction time at neutral to alkaline pH.
Enzymes structurally classified as members of family 7 of the carbohydrate esterases (CE-7) have been employed as perhydrolases to catalyze the reaction of hydrogen peroxide (or alternative peroxide reagent) with alkyl esters of carboxylic acids in water at a basic to acidic pH range (from ca. pH 10 to ca. pH 5) to produce an efficacious concentration of a peroxycarboxylic acid for such applications as disinfection (such as medical instruments, hard surfaces, textiles), bleaching (such as wood pulp or paper pulp processing/delignification, textile bleaching and laundry care applications), and other laundry care applications such as destaining, deodorizing, and sanitization (Published U.S. Patent Application Nos. 2008-0176783, 2008-0176299, 2009-0005590, and 2010-0041752 to DiCosimo et al.). The CE-7 enzymes have been found to have high specific activity for perhydrolysis of esters, particularly acetyl esters of alcohols, diols and glycerols. Published U.S. Patent Application No. 2010-0087529 to DiCosimo et al. describes several variant CE-7 perhydrolases derived from several Thermotoga sp. having higher perhydrolytic specific activity and/or improved selectivity for perhydrolysis when used to prepare peroxycarboxylic acid from carboxylic acid esters. One of the variants described in Published U.S. Patent Application No. 2010-0087529, Thermotoga maritima C277S, exhibited a significant improvement in specific activity relative to the T. maritima wild-type enzyme.
Recombinant microbial production of an enzyme often includes one or more downstream biomass processing steps used to partially or completely purify and/or concentrate the recombinant enzyme from other components of the biomass. However, considerable difficulty has been encountered when filtering or concentrating protein preparations on microfiltration membranes. The purpose of the filtration is to pass a solution containing the perhydrolase through the membranes while retaining particles greater than 0.2 microns or 0.45 microns in the retentate. The protein is often retained, at least in part, by the membrane, rather than passing through it, where the porosity of the membrane is such that the protein should freely pass through it.
One of the biomass components that may be adversely impacting the ability to recovery and/or concentrate the desired enzyme catalyst is the presence of DNA in the cell homogenate. However, the addition of an exogenous deoxyribonuclease (DNAse) may not be cost effective, especially for industrial scale fermentations, and the use of mammalian sources of DNAse may be undesirable in toll fermentation.
The problem to be solved is to provide a facile and cost-effective process to obtain a concentrate comprising a recombinant enzyme having perhydrolytic activity, preferably a process that does not include the use of an exogenous deoxyribonuclease.